JP2012527990A - How to protect the membrane - Google Patents

Abstract

The present invention relates to a method for protecting a membrane by treatment with an aqueous solution containing at least one water-soluble / nucleophilic compound and to the use of this aqueous solution for protecting a matrix.

Description

  The present invention relates to methods and uses for protecting a matrix, such as a membrane, and more particularly a silica membrane. The device and use is suitable for use in, for example, biochemistry, molecular biology, molecular genetics, microbiology, medical diagnostics, or forensic medicine.

  Matrixes, and more particularly membranes such as silica membranes, are widely used in the fields of biochemistry, molecular biology, molecular genetics, microbiology, medical diagnostics, or forensic medicine and usually purify / single biomolecules. Used to release. A frequently used method is, for example, use in the isolation of nucleic acids such as DNA or RNA.

  For this purpose, the sample containing the DNA and / or RNA to be isolated is bound to a (purified) matrix, for example in the presence of a “chaotropic” reagent. The other components of the sample are then removed by rinsing and washing. The DNA or RNA is then released and analyzed.

  As part of the in-house study by Applicants, there are now some matrices, and more particularly, commercially available matrices, in some cases, especially when they are present in the form of membranes, in some cases with nucleic acids along with (storage) time. It has been shown that this creates the problem of reduced ability to bind. This is especially true when the matrices are stored at room temperature or above. This problem can be suppressed by storing at 4 ° C, but cannot be completely prevented.

  The object of the present invention is to at least effectively overcome the above-mentioned drawbacks apparent in the prior art, and more particularly to a method and use capable of protecting a matrix from aging in a wide range of applications. Is to create.

  The above objective is accomplished by a method according to claim 1 of the present invention. In this way, a method for protecting a film by treatment with an aqueous solution containing at least one water-soluble / nucleophilic compound is proposed.

  The above object is likewise achieved by the use according to claim 2 of the present invention. Thus, the use of an aqueous solution containing at least one water-soluble / nucleophilic compound for protecting the membrane is proposed.

  For the purposes of the present invention, the term “aging” of a matrix, in particular a membrane, for example, is understood to mean that the nucleic acid loses the ability to bind to a suitable matrix under chaotropic conditions. The inventors suspect that this is due to the long-term storage of the matrix in the presence of various plastics or in the form of a simple integrated spin column. This can lead to outgassing of plastic components such as plasticizers, or other additives and / or styrene, or short chain aliphatic compounds. In extreme cases, this result induces complete hydrophobicity of the matrix, which can be associated with a rapid drop in yield in various nucleic acid processing protocols. This is because there is a great possibility that these outgassings bind to the hydrophilic surface of the matrix.

  For the purposes of the present invention, the term “nucleic acid” is not particularly limited, but in particular a natural, preferably linear, branched or circular nucleic acid, eg RNA, more particularly Is mRNA, single- and double-stranded viral RNA, siRNA, miRNA, snRNA, tRNA, hnRNA, ribozyme, genomic, bacterial, or viral DNA (single- and double-stranded), chromosome and episomal DNA, free circulation Synthetic or modified nucleic acids such as nucleic acids such as plasmids or oligonucleotides, more particularly primers, probes or standard reagents used for PCR, nucleic acids labeled with digoxigenin, biotin or fluorescent dyes, or LNA (Locked nucleic acid) or PNA (peptide nucleic acid) is understood to mean.

  The term “matrix” is understood to mean, but is not limited to, in particular, a solid phase capable of reversibly binding to biomolecules, preferably nucleic acids. For the purposes of the present invention, such a solid phase is preferably a membrane, particularly preferably a silica membrane. However, the matrix for the purposes of the present invention is further a filter material comprising a mineral component such as a metal oxide, more particularly aluminum oxide, nitride, carbide, more particularly silicon carbide. Or a hydrophilic particle capable of forming a loose or tight packing.

  For the purposes of the present invention, the term “immobilization” is understood to mean in particular, but not limited to, reversible immobilization in a suitable solid phase.

  The term “nucleophilic” refers to the ability of a negative polarity molecule (“nucleophile”) to attack a positively charged or positively charged atom in the molecule to form a covalent bond. It is understood. Typical nucleophiles are often negatively charged or have at least one free electron pair in a high energy orbit.

  In a preferred embodiment, the water-soluble nucleophilic compound according to the invention is a negatively charged detergent and / or has at least one molecule with at least two OH groups.

  The process according to the invention and / or the use according to the invention comprises treatment with an aqueous solution comprising at least one water-soluble nucleophilic compound. This is because such compounds have the same chemical properties as the solid phase itself, and are therefore most likely to “mimic” the surface of the solid phase, eg, the silica membrane surface.

  The reason for the surprising effects of the method and / or use of the present invention remains unclear so far. However, the inventors believe that the method and / or use of the present invention may result in the aforementioned capture of outgassing of the plastic component which is responsible for the aging of the matrix. The inventors think that the water-soluble / nucleophilic compound may bind to the gas release of the plastic component. In this way, it is likely that the most attacked by outgassing is not the matrix but the water-soluble, nucleophilic compound according to the invention.

Such methods and / or uses provide at least one of the following advantages for a wide range of applications within the context of the present invention:
The solid phase or matrix is protected by simple and very rapid manipulation. This is because the treatment simply involves soaking the matrix in the aqueous solution according to the invention.
The method and / or use of the invention is preferably protective (matrix impregnation), ie the matrix is impregnated before storage. This therefore means that no further operation is required at the end user.
-Impregnation ensures constant quality and performance on the side of the matrix.
-In many applications within the context of the present invention, the protection is so complete that storage at low temperatures can be dispensed with.
As a result, the reproducibility in use is significantly increased.

  In a preferred embodiment of the invention, the matrix is hydrophilic. In a particularly preferred embodiment of the invention, the matrix is a hydrophilic membrane. “Boom treatment” (EP819696) shows an example of nucleic acid binding to a hydrophilic membrane utilizing a silica membrane. For selective binding of nucleic acids, the sample is lysed in a lysis buffer containing a chaotropic substance, such as guanidinium thiocyanate. Not only are cells lysed, but proteins are denatured and inactivated. The nucleic acid is released and binds to the OH group of the silica membrane. The other components of the sample can then be removed by washing. Finally, DNA or RNA can be released for subsequent analysis.

  Accordingly, preferred hydrophilic membranes are silica membranes, also known as silica membranes, glass fiber filters, quartz wool, or glass wool, as well as filter membranes that may or may not have functional groups, Or a filter membrane composed of a synthetic organic polymer such as regenerated cellulose, cellulose acetate, nitrocellulose, polyamide, or poly (ether) sulfone.

  For example, the nucleic acid is bonded to the silica surface via a hydrogen bond with the Si—OH group (silanol group) of the silica film. The aforementioned released gas of the plastic component will probably also bind to these Si-OH groups, thus resulting in the hydrophobicity of the matrix. Similar to the case of bonding to the silica film via the Si—OH group, the water-soluble compound according to the present invention can provide an electron pair for forming a covalent bond based on its nucleophilicity. We believe that treatment with the aqueous solution of the present invention may result in the compound according to the present invention being attacked by outgassing of the plastic component instead of the matrix surface.

In a preferred embodiment of the invention, the OH group according to the invention is an alcoholic OH group. OH as classical Lewis base ^- is nucleophilic. It has free electron pairs that can be supplied for coupling.

  In a preferred embodiment of the invention, the membrane is used for nucleic acid immobilization.

  In a preferred embodiment, the method according to the invention is a method for impregnation and / or the use according to the invention is a use for impregnation. The membrane is treated with an aqueous solution according to the invention before storage, so that it is prevented from the aging described above.

  In yet another preferred embodiment, the method according to the invention and / or the use according to the invention comprises a drying step after treatment with an aqueous solution. This drying step is preferably performed at a temperature of ≧ 5 ° C. to ≦ 45 ° C., 20 ° C., 21 ° C., 22 ° C., 23 ° C., 24 ° C., 25 ° C., 26 ° C., 27 ° C., 28 ° C., 29 ° C. Or a temperature of 30 ° C. is preferred. In principle, there is no upper or lower limit in the temperature range, but temperatures up to 45 ° C. are preferred for operational reasons. The duration of the drying process is preferably between ≧ 1 second and ≦ 60 minutes, but in principle there is no upper or lower limit. For operational or production reasons, 1 minute, 2 minutes, 3 minutes, 4 minutes, or up to 5 minutes are preferred. This drying step is advantageous for operational reasons, for example in the case of spin column integration pretreatment and / or for storage reasons.

  In a preferred embodiment, the water-soluble / nucleophilic compound according to the invention is a solid. Therefore, the water-soluble / nucleophilic compound can remain on the surface of the matrix as a thin impregnated layer until the time of use after the drying step. Further, since the nucleophilic compound is water-soluble, a separate washing step for removing the water-soluble / nucleophilic compound is not necessary. This is because this compound is dissolved upon contact with the nucleic acid sample and can therefore be removed in the usual washing step of the nucleic acid purification procedure.

  In a preferred embodiment, the water-soluble / nucleophilic compound according to the invention having at least one molecule with at least two OH groups is a sugar alcohol. In a particularly preferred embodiment, the water-soluble nucleophilic compound according to the invention having at least one molecule with at least two OH groups is sorbitol, xylitol, lactitol, threitol, erythritol, mannitol, isomalt, inositol, palmitate And / or citrate, or a mixture thereof.

  In yet another particularly preferred embodiment, the aqueous solution according to the invention comprises at least one negatively charged detergent and at least one water-soluble / nucleophilic compound having at least one molecule with at least two OH groups. At least one mixture.

  In yet another particularly preferred embodiment, the negatively charged detergent is a fatty alcohol sulfate, more particularly sodium dodecyl sulfate (SDS) and / or alkylbenzene sulfonic acid and / or alkylbenzene sulfonate. More particularly selected from the group comprising sodium dodecylbenzenesulfonate, benzenesulfonate, dodecylbenzenesulfonate, ammonium dodecylbenzenesulfonate, and / or N-lauroyl sarcosine ("sarcosyl"), or mixtures thereof It is.

  As already revealed, the water-soluble compounds according to the invention have a much higher probability of being able to “mimic” the matrix surface due to their nucleophilicity. Therefore, perhaps the compound according to the invention, rather than the matrix surface, is under attack by the outgassing of the plastic component.

  In a preferred embodiment, the aqueous solution according to the invention further comprises a compound that inhibits the growth of microorganisms. Particularly preferably, this compound is selected from the group comprising sodium azide, thimerosal, phenol, benzyl alcohol and / or cresol.

  The treatment with the aqueous solution of the invention preferably lasts from ≧ 1 second to ≦ 60 seconds. In principle, the duration of the treatment has no upper limit, but in many applications it has been found that treatment longer than 5 minutes does not result in a substantial improvement in binding capacity. Thus, the preferred duration of treatment is 1 minute, 2 minutes, 3 minutes, 4 minutes, up to 5 minutes.

  Further, the treatment with the aqueous solution of the present invention is preferably performed at a temperature of ≧ 5 ° C. to ≦ 45 ° C., 20 ° C., 21 ° C., 22 ° C., 23 ° C., 24 ° C., 25 ° C., 26 ° C., 27 ° C., A temperature of 28 ° C, 29 ° C or 30 ° C is preferred. In principle, there is no upper or lower limit in the temperature range, but temperatures up to 45 ° C. are preferred for operational reasons.

  Furthermore, the pH of the aqueous solution of the present invention is preferably ≧ 4.5 to ≦ 9.5, particularly preferably ≧ 6 to ≦ 8, even more preferably about 7. In other words, the pH of the aqueous solution of the present invention is most preferably neutral in nature.

  Furthermore, the aqueous solution of the present invention preferably has a concentration of ≧ 0.5 to ≦ 20%, particularly preferably a concentration of ≧ 1 to ≦ 10%, and even more preferably a concentration of ≧ 1 to ≦ 5%.

  The components described above, claimed and described in the exemplary embodiments, used in accordance with the present invention are constrained to any particular exceptional conditions in their size, shape, material selection, and technical concepts. Therefore, selection criteria known in the application field can be applied without limitation.

  Further details, features and advantages of the subject-matter of the invention are given in the dependent claims and also by way of example, the following description, the attached drawings and exemplary embodiments, ie a plurality of the invention. Possible embodiments and uses of this will be apparent from the embodiments specifically shown.

An experimental setup for inducing membrane aging is shown. 2 shows a graph of plasmid DNA binding ability of a silica membrane pretreated according to the present invention and a comparative example. Figure 6 shows a graph of plasmid DNA binding capacity of silica membranes pretreated with various concentrations of sorbitol or SDS, and comparative examples. 2 shows a graph of RNA binding capacity of silica membranes pretreated according to the present invention and comparative examples. Figure 3 shows a graph of RNA binding capacity of silica membranes pre-treated with various concentrations of sorbitol or SDS and comparative examples.

  The present invention is specifically illustrated by the following exemplary embodiments, but should not be limited thereto.

The following procedure was used:
Example 1
Silica membrane disks (GF51, Pall) were cut out with a punch and each was immersed in an aqueous solution of the corresponding substance. Corresponding substances (used) will be described later. Immersion was performed at room temperature, ie, about 20 ° C. for 5 minutes. The membrane disc was then dried briefly and incubated at 50 ° C. in the presence of a relatively large number of frit (Vyon F polyethylene, Koepp). This incubation induced aging of the membrane disk. Incubation lasted 7 days (Figures 2 and 4) or 3 weeks (Figures 3 and 5). The experimental setup for this purpose is shown in FIG. Fill a closed beaker (1) with a number of frits (3). Next, an open aluminum boat containing the treated membrane disk (2) is placed on the frit.

  The membrane disc was then mounted directly on a Mini spin column (setup from bottom to top: frit / 1 × GF51 membrane / clamp ring) prior to testing (for binding capacity).

  For DNA testing, 10 μg of pUC21 plasmid was dissolved in 500 μl of buffer PB (QIAGEN), applied to the column and centrifuged through the membrane. Then, it was washed with 700 μl of buffer PE (QIAGEN), dried and centrifuged, and eluted with 200 ml of buffer EB (QIAGEN). The eluate was measured optically at 260 nm.

  The “recovery rate” of the silica membrane can be found in Table 1 and FIG. Shown in each case is the binding capability of membrane discs as obtained, untreated membrane discs, and membrane discs pretreated with sorbitol, NaCl, SDS, or cetyltrimethylammonium bromide (CTAB).

  The results showed that the untreated membrane was highly hydrophobic and virtually lost its binding ability. Treatment with NaCl or the cationic detergent CTAB also did not provide any improvement.

  The membrane pretreated with sorbitol and / or SDS according to the present invention surprisingly retained its binding ability and did not show aging effects.

  In another experiment (not shown in FIG. 2), PEG 600 and PEG 4000 were similarly tested. For both materials, the membrane remained hydrophilic unlike the untreated membrane, but nevertheless showed no improvement in binding capacity after storage.

  The binding ability of silica membranes pretreated with various concentrations (1%, 5%, or 10%) of sorbitol or SDS can be found in Table 2 and FIG.

  The results showed that the untreated membrane disk and the membrane disk soaked in water lost virtually all of their binding capacity. In contrast, all sorbitol or SDS impregnated membranes were within the range of reference samples that were not stored.

Example 2
The silica film disk was pretreated as described in Example 1 and mounted on a Mini spin column.

In the test, 5 × 10 ⁵ HeLa cells per preparation were homogenized in 350 μl buffer RLT, mixed with the same amount of 70% strength ethanol, applied to a prepared spin column and centrifuged through the membrane. Spin columns were processed according to standard RNeasy procedures (QIAGEN RNeasy Mini handbook; protocol: purification of total RNA from animal cells by spin technique). Elution was performed in 30 μl RNase-free water. The eluate was measured optically at 260 nm.

  The “recovery rate” of the silica membrane can be found in Table 3 and FIG. Shown in each example are a standard RNeasy column (QIAGEN), a fresh silica membrane, an untreated silica membrane, and silica pretreated with sorbitol, NaCl, SDS, or cetyltrimethylammonium bromide (CTAB) This is the RNA binding ability of the membrane.

  Surprisingly, as in Example 1, the membrane treated with SDS and / or sorbitol according to the invention did not show any aging effect in this example either.

  In contrast, treatment with NaCl showed no noticeable improvement, and treatment with CTAB resulted in a significant decrease in yield.

  The RNA binding capacity of silica membranes pretreated with various concentrations (1%, 5%, or 10%) of sorbitol or SDS can be found in Table 4 and FIG.

  All membranes soaked in sorbitol and / or SDS according to the present invention were within the range of reference membranes that were not preserved.

Claims (18)

A method for protecting a membrane by treatment with an aqueous solution containing at least one water-soluble / nucleophilic compound. Use of an aqueous solution containing at least one water-soluble and nucleophilic compound to protect the membrane. 3. The method and / or claim 2 wherein the water soluble / nucleophilic compound is a negatively charged detergent and / or has at least one molecule with at least two OH groups. Use as described in. A method and / or use according to any preceding claim, wherein the OH group is an alcoholic OH group. Method and / or use according to any of the preceding claims, characterized in that the membrane is used for immobilization of nucleic acids. A method and / or use according to any of the preceding claims, wherein the method is a method for impregnation and / or the use is a use for impregnation. A method and / or use according to any preceding claim, characterized in that the method and / or the use comprises a drying step after treatment with an aqueous solution. The method and / or use according to any preceding claim, wherein the water-soluble / nucleophilic compound is a solid. The method and / or use according to any preceding claim, wherein the water-soluble and nucleophilic compound having at least one molecule having at least two OH groups is a sugar alcohol. The water-soluble / nucleophilic compound having at least one molecule having at least two OH groups is sorbitol, xylitol, lactitol, threitol, erythritol, mannitol, isomalt, inositol, palmitate, and / or citrate, Or a method and / or use according to claim 9, characterized in that it is selected from or a mixture thereof. The negatively charged detergent is a fatty alcohol sulfate, more particularly sodium dodecyl sulfate (SDS) and / or alkylbenzene sulfonic acid and / or alkyl benzene sulfonate, more particularly dodecyl benzene sulfonic acid. Any of the previous claims, characterized in that it is selected from the group comprising sodium, benzene sulfonic acid, dodecyl benzene sulfonate, ammonium dodecyl benzene sulfonate, and / or N-lauroyl sarcosine, or mixtures thereof. Method and / or use. The aqueous solution contains at least one mixture of at least one negatively charged detergent and at least one water-soluble / nucleophilic compound having at least one molecule having at least two OH groups. A method and / or use according to any of the preceding claims. The method and / or use according to any preceding claim, wherein the aqueous solution additionally comprises a compound that inhibits the growth of microorganisms. 14. Method and / or use according to claim 13, characterized in that said additional compound is selected from the group comprising sodium azide, thimerosal, phenol, benzyl alcohol and / or cresol. A method and / or use according to any preceding claim, wherein the treatment lasts from ≧ 1 second to ≦ 60 seconds. A method and / or use according to any preceding claim, wherein the treatment is carried out at a temperature of ≧ 5 ° C to ≦ 45 ° C. A method and / or use according to any preceding claim, wherein the treatment is carried out at a pH of ≧ 4.5 to ≦ 9.5. A method and / or use according to any preceding claim, wherein the aqueous solution has a concentration of ≧ 0.5 to ≦ 20%.

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